MicroFuelPro: Microbial fuel development framework using synthetic biology for next generation drop-in renewable fuel production

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The employment of bacteria as cell factories is an attractive means for sustainable large production of energy molecules. Bioengineering research have made impressive progresses in identifying and optimizing microbial metabolic pathways involved in the biosynthesis of fuel-like hydrocarbons. Such metabolic routes include: derivations of amino acid pathway, the mevalonate pathway, the polyketide pathway, and the fatty acid pathway. These natural metabolic routes have been engineered and modestly implemented in native and non-native hosts, enabling the microbial cell to assimilate simple sugars into value-added molecules. However, industrial production rate has not been achieved yet, moreover, this biosynthesis cannot be considered entirely sustainable, unless it is decoupled from the use of simple sugars as feedstock. MicroFuelPro project sets the challenge of enhancing metabolic fluxes to achieve large scale biofuel production through the microbial assimilation of waste material, such as lignocelluloses, into energy molecules. The direct conversion of waste materials into fuel-like molecules has not been fully explored, thus, the objective of the project will lies in the identification of genes that enable this catabolic and anabolic process efficiently. An attractive candidate to host biofuel synthesis is the Gram-negative bacterium Zymomonas mobilis, which has a high substrate uptake and striking ethanol yield. However, Z. mobilis does not naturally degrade lignocellulose, hence, to provide it with this function, relevant genes will be identified in lignocellulolytic microorganisms and expressed in the host platform. Most likely, the solely heterologous expression of genes will not be sufficient to enable an efficient substrate assimilation, thus, further bioengineering tools (e.g. CRISPR, genome editing, ribosome binding molecules etc.) will be used to generate a synthetic high-performance metabolism. The same approach will be exerted in other different bacteria to synthesize compounds such as fatty acids, terpenoids, polyketides and higher alcohols. The pathways involved in these products synthesis will be either tweaked in native host or heterogously engineered in model bacteria that show features like high growth rate, high tolerance to solvent toxicity, or ability of metabolizing alternative feedstocks. The ultimate goal will be the comparison of an array of engineered bacteria to detect the most suitable candidates for lignocellulose biodegradation and conversion into value-added molecules. After the production of fuel molecules, the fuel design properties will be identified, and surrogate fuel pallet will be chosen. Ideally, each palette compound would be representative of a class of compounds found in the target renewable fuel. The surrogate palette will contain representatives from each of the major hydrocarbon families found in market hydrocarbon fuels: n-alkanes, iso-alkanes, cycloalkanes, aromatics, and naphtho-aromatics. 13C (carbon) and 1H (proton) nuclear magnetic resonance (NMR) spectroscopy and GC-MS will be used to quantify the compositional characteristics of each target renewable fuel in this study. The next step will be to identify and run an optimization study to determine how much of each palette compound should be included in the surrogate to achieve the property targets of renewable fuels. Once each surrogate composition is determined, the pure palette compounds will be blended together to produce the surrogates, and each surrogate will be tested to determine whether the property targets are achieved within their desired tolerances. The outcomes of the fuel design process will be correlated with the metabolic pathways. Through this analysis we will be able to identify and design the most efficient pathway not only in terms of yield but also in terms of fuel properties, sustainability and suitability to conventional and future power generation systems.